ونحن ممتنون لويلكوم ترست والمنسق للدعم المالي. IMGG هو زميل EMBO. KLH وهو طالب دكتوراه BBSRC التي تمولها. نشكر فيليب روبين وTidball باتريك لدعم الثقافة الفنية والخلية، والدكتور أندرو دوهرتي للصيانة والمساعدة مع المجاهر.

<ol>
	<li>Sankaranarayanan, S., De Angelis, D., Rothman, J. E., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pHluorins+for+optical+measurements+of+presynaptic+activity.">The use of pHluorins for optical measurements of presynaptic activity.</a> Biophys. J. 79, 2199-2208 (2000).</li><li>Ashby, M. C., Ibaraki, K., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=It's+green+outside:+tracking+cell+surface+proteins+with+pH-sensitive+GFP.">It's green outside: tracking cell surface proteins with pH-sensitive GFP.</a> Trends Neurosci. 27, 257-261 (2004).</li><li>Swaminathan, R., Hoang, C. P., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+recovery+and+anisotropy+decay+of+green+fluorescent+protein+GFP-S65T+in+solution+and+cells:+cytoplasmic+viscosity+probed+by+green+fluorescent+protein+translational+and+rotational+diffusion.">Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion.</a> Biophys. J. 72, 1900-1907 (1997).</li><li>Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+green+fluorescent+protein+and+its+mutants+in+quantitative+fluorescence+microscopy.">Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.</a> Biophys. J. 73, 2782-2790 (1997).</li><li>Wiedenmann, J., Oswald, F., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+for+live+cell+imaging:+opportunities,+limitations,+and+challenges.">Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges.</a> IUBMB Life. 61, 1029-1042 (2009).</li><li>Ashby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+AMPA+receptors+(AMPARs)+from+synapses+is+preceded+by+transient+endocytosis+of+extrasynaptic+AMPARs.">Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs.</a> J. Neurosci. 24, 5172-5176 (2004).</li><li>Bouschet, T., Martin, S., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-activity-modifying+proteins+are+required+for+forward+trafficking+of+the+calcium-sensing+receptor+to+the+plasma+membrane.">Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane.</a> J. Cell. Sci. 118, 4709-4720 (2005).</li><li>Ashby, M. C., Maier, S. R., Nishimune, A., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+diffusion+drives+constitutive+exchange+of+AMPA+receptors+at+dendritic+spines+and+is+regulated+by+spine+morphology.">Lateral diffusion drives constitutive exchange of AMPA receptors at dendritic spines and is regulated by spine morphology.</a> J. Neurosci. 26, 7046-7055 (2006).</li><li>Martin, S., Bouschet, T., Jenkins, E. L., Nishimune, A., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+regulation+of+kainate+receptor+surface+expression+in+hippocampal+neurons.">Bidirectional regulation of kainate receptor surface expression in hippocampal neurons.</a> J. Biol. Chem. 283, 36435-36440 (2008).</li><li>Jaskolski, F., Mayo-Martin, B., Jane, D., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-dependent+membrane+drift+recruits+AMPA+receptors+to+dendritic+spines.">Dynamin-dependent membrane drift recruits AMPA receptors to dendritic spines.</a> J. Biol. Chem. 284, 12491-12503 (2009).</li></ol>

الإعلان عن أي تضارب في المصالح.

وصفنا ووضع استراتيجية مبتكرة لتصور مكونات غشاء البلازما الاتجار البروتين. النهج التوافقي من photobleaching التقنيات مع بروتين سبتمبر الموسومة تمكن بشكل انتقائي على أن يقسم الأحداث البلازما الإدراج غشاء. بواسطة photobleaching باستمرار بروتينات غشاء في المناطق المحيطة أثناء الانتعاش، وهي طريقة &quot;FRAP-FLIP &#39;يقيم مساهمة الاتجار حويصلي إلى انتعاش مضان. هذا النهج الجديد يسمح للتسجيل المباشر لإدخال غشاء من البروتين، وتمكين كل من عدد من الغرف الفرعية حيث لوحظ انتعاش واتساع الانتعاش (ΔF) في حالة مستقرة يحدد لاحقا. كذلك، مقارنة مع FRAP ودون FLIP يسمح نسبة الانتعاش يعزى إلى انتشار الأفقي يتم حسابها. وعلاوة على ذلك، في التجربة ذاتها، ويمكن مناطق غير photobleached التغصنات المتاخمة للمناطق المحيطة photobleached تكونتقييم نوعيا خلال الانتعاش، وفقدان مضان لوحظ في هذه المناطق سوف يكون راجعا إلى نشر الوحشي للمستقبلات photobleached في هذه القطاعات غير photobleached من التغصنات. ويمكن استخدام هذا البروتوكول انتقائي photobleaching للتحقيق في مجموعة متنوعة من العمليات الخلوية مثل الاتجار تميز excocytosis في غشاء البلازما في المناطق الفرعية محددة (على سبيل المثال التشعبات أو العمود الفقري،) FRAP أو تقييم مساهمة الجانبي نشر الإدراج مقابل عن طريق إجراء FRAP متوازية و&#39; -FLIP &#39;البروتوكولات على طول التشعبات المجاورة (الشكل 3). بوضوح، في حين تم تقديم مبادئ توجيهية محددة، وسوف تحتاج إلى كل مختبر تحسين المعلمات التصوير وفقا لنماذج محددة والمعدات. الأهم من ذلك، كل المستقبلات السطحية في العائد على الاستثمار تحتاج إلى أن تكون photobleached، مستقلة عن الطائرة Z-محور التركيز، ولكن دون وقوع خسائر كبيرة الضوئية أو photobleaching غير محددة. لناوينبغي توخي الحذر المتطلبات البيئية عند محاولة هذا البروتوكول في وسوما الخلية، حيث أن نسبة عالية من مقصورات درجة الحموضة منخفضة نسبيا داخل الخلايا عادة النتائج في مضان خلفية عالية في هذه المناطق. وعلاوة على ذلك، في حين أننا أظهرنا كيف يمكن تطبيق هذه التقنية في تختلف طرق، قبل البدء انتقائية التجارب photobleaching على جديد سبتمبر الموسومة بناء، فإننا ننصح أن تتم أولا إلى توصيف الأولي للمستقبلات المفتاحية، كما وصفها آشبي وآخرون 2. وعموما، هذا الأسلوب هو التكيف مع قوة وتنوعا من بروتوكول FRAP القياسية، تمكن أحداث الإدراج في غشاء البلازما التي يتم تقييمها في الوقت الحقيقي القريب.

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; اسم كاشف</strong</td&gt;<td<strong&gt; شركة</strong</td&gt;<td<strong&gt; فهرس العدد</strong</td&gt;<td<strong&gt; تعليقات (اختياري)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; coverslips زجاج 24MM</td&gt;<td&gt; VWR الدولية</td&gt;<td&gt; 631-0161</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; بولي-L-يسين</td&gt;<td&gt; سيجما</td&gt;<td&gt; P2636</td&gt;<td&gt; 1mg/ml في عازلة بورات إلى coverslips معطف</td&gt;</tr&gt;<tr&gt;<td&gt; متوسطة Neurobasal</td&gt;<td&gt; إينفيتروجن</td&gt;<td&gt; 21103</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; B27</td&gt;<td&gt; إينفيتروجن</td&gt;<td&gt; 17504-044</td&gt;<td&gt; 2٪ في تصفيح العصبية وتغذية المتوسطة</td&gt;</tr&gt;<tr&gt;<td&gt; البنسلين الستربتوميسين</td&gt;<td&gt; سيجما</td&gt;<td&gt; P0781</td&gt;<td&gt; متوسط ​​1٪ في تصفيح العصبية وتغذية</td&gt;</tr&gt;<tr&gt;<td&gt; L-الجلوتامين</td&gt;<td&gt; إينفيتروجن</td&gt;<td&gt; 25030</td&gt;<td&gt; 2MM / 0.8mM في طلاء / تغذية المتوسطة</td&gt;</tr&gt;<tr&gt;<td&gt; حصان المصل</td&gt;<td&gt; Biosera</td&gt;<td&gt; DH-291H</td&gt;<td&gt; 10٪ في وسائل الاعلام طلاء الخلايا العصبية فقط</td&gt;</tr&gt;<tr&gt;<td&gt; pSIN ناقلات الاستنساخ ReP5</td&gt;<td&gt; إينفيتروجن</td&gt;<td&gt; K75001</td&gt;<td&gt; للحصول على إنتاج فيروس سندبيس</td&gt;</tr&gt;<tr&gt;<td&gt; LSM510 نظام META مبائر</td&gt;<td&gt; زييس</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; صورة J برامج</td&gt;<td&gt; المعاهد الوطنية للصحة</td&gt;<td&gt;</td&gt;<td&gt; برامج الوصول المفتوحة. جميع الإضافات الموصوفة هنا متاحة في<a href="http://rsbweb.nih.gov/ij/plugins/"&gt; http://rsbweb.nih.gov/ij/plugins/</a</td&gt;</tr&gt;</tbody&gt;</table

lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching

<p class="jove_content"بروتينات الغشاء&gt; مثل المستقبلات والقنوات الأيونية يخضع الاتجار نشط في الخلايا العصبية، والتي هي مستقطبة للغاية ومعقدة شكليا. هذا الاتجار الموجهة له أهمية أساسية لتقديم وصيانة أو إزالة البروتينات متشابك.</p&gt;<p class="jove_content"&gt; سوبر مسير الشمس pHluorin (سبتمبر) هو مشتق درجة الحموضة حساسة من eGFP التي استخدمت على نطاق واسع لتصوير الخلايا الحية للبروتينات غشاء البلازما<sup&gt; 1-2</sup&gt;. في انخفاض الرقم الهيدروجيني، بروتوناتيون من سبتمبر يقلل امتصاص الفوتون ويزيل الانبعاثات مضان. كما أحداث الاتجار معظم الخلايا يحدث في مقصورات مع انخفاض الرقم الهيدروجيني، حيث طغت سبتمبر مضان، في إشارة مضان من سبتمبر الموسومة البروتينات هي في الغالب من غشاء البلازما التي تعرض سبتمبر في بيئة درجة الحموضة محايد خارج الخلية. عندما تضاء في سبتمبر الكثافة العالية، مثل كل صبغة الفلورسنت، هو photodamaged لا رجعة فيه (photobleached)<sup&gt; 3-5</sup&gt;. الأهم من ذلك، أعرب عن سطح فقط بسبب انخفاض الرقم الهيدروجيني يروي امتصاص الفوتون، يمكن photobleached سبتمبر في حين سبتمبر الخلايا لا تتأثر الإضاءة عالية الكثافة<sup&gt; 6-10</sup&gt;.</p&gt;<p class="jove_content"&gt; FRAP (استرداد مضان بعد photobleaching) من سبتمبر الموسومة البروتينات هي تقنية مريحة وقوية لتقييم ديناميات البروتين في غشاء البلازما. عندما يتم photobleached البروتينات الموسومة fluorescently في المنطقة ذات الاهتمام (ROI) الانتعاش في مضان يحدث نتيجة لحركة غير مقصور البروتينات سبتمبر الموسومة في المنطقة المبيض. وهذا يمكن أن يحدث عن طريق نشر الجانبية و / أو من الإيماس غير photobleached مستقبلات الموردة إما عن طريق<em&gt; من جديد</em&gt; تخليق أو إعادة التدوير (انظر الشكل 1). يمكن تحديد جزء من بروتين غير متحركة ومتنقلة، ويمكن استجوابه في التنقل والحركية للجزء قابل للانتشار في ظل ظروف القاعدية وحفزت مثل تطبيق ناهض أو محفزات تنشيط الخلايا العصبية مثل NMDA أو تطبيق بوكل<sup&gt; 8،10</sup&gt;.</p&gt;<p class="jove_content"&gt; نحن وصف تقنيات photobleaching تهدف إلى تصور انتقائي استعادة مضان تعزى إلى الإيماس. لفترة وجيزة، وعائد استثمار photobleached مرة واحدة كما هو الحال مع بروتوكولات FRAP قياسي، ثم، بعد انتعاش قصير، عن طريق تكرار تبييض المناطق المحيطة. وقد استخدم هذا البروتوكول &quot;FRAP-FLIP &#39;، التي وضعت في المختبر، لتوصيف أمبا الاتجار مستقبلات في العمود الفقري شجيري<sup&gt; 10</sup&gt;، وقابلة للتطبيق على مجموعة واسعة من الدراسات لتقييم الاتجار الاتجار داخل والإيماس.</p

Watch this Scientific Journal Video about Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching at JoVE.co

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

هذا التقرير وصفا للاستخدام التصوير الخلية الحية وتقنيات photobleach لتحديد التعبير السطح، ومسارات النقل وحركية الاتجار خارجيا أعرب GFP الموسومة درجة الحموضة حساسة، والبروتينات في الغشاء البلازمي للخلايا العصبية.

المقررة الإنتشار الأفقي والإيماس من غشاء بروتينات في الخلايا العصبية مثقف باستخدام الاسترداد الإسفار وPhotobleaching الإسفار الخسارة

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

lateral-diffusion-exocytosis-membrane-proteins-cultured-neurons

Research

JoVE Journal

Neuroscience

82.9K Views.  University of Bristol. هذا التقرير وصفا للاستخدام التصوير الخلية الحية وتقنيات photobleach لتحديد التعبير السطح، ومسارات النقل وحركية الاتجار خارجيا أعرب GFP الموسومة درجة الحموضة حساسة، والبروتينات في الغشاء البلازمي للخلايا العصبية.

Video: Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

مضان الانتعاش بعد Photobleaching (FRAP) من البروتينات المرتبطة دلاليا الإسفار في العمود الفقري شجيري الخلايا العصبية الحصين مثقف

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

تحديد أنواع غشاء الميتوكوندريا المحتملة والأوكسجين التفاعلي في الخلايا العصبية الفئران الحية القشرية

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

تحليل الصرف العمود الفقري شجيري الخلايا العصبية في الجهاز العصبي المركزي لؤلؤ

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

تحليل كمي متشابك الحويصلة التجديد حمام سباحة في الخلايا العصبية مثقف الحبيبية للدماغ باستخدام الأصباغ FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

حفز النمو في الخلايا العصبية شجيري متعاطف مثقف

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

التصوير pHluorin الموسومة الإدراج مستقبلات غشاء البلازما إلى الخلايا العصبية في الابتدائية الماوس مثقف

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

وصفها الفرق من سطح الخلية والمنضوية البروتينات الأجسام المضادة بعد تغذية الخلايا العصبية مثقف لايف

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

إعداد متشابك غشاء البلازما والبروتينات الكثافة بعد المشبكي باستخدام التدرج السكروز متقطع

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

مضان وتلألؤ بيولوجي التصوير من التحت خلوية كا<sup&gt; 2+</sup&gt; في المسنين الحصين الخلايا العصبية

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

كمي من إندوسوم و ليسوسوم التحركات في الخلايا العصبية المستزرعة باستخدام تحقيقات الفلورسنت

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...